PROJECT SUMMARY Immunological memory is the ability of our immune system to respond with greater strength and quickness upon re-encounter with the same pathogen (i.e. secondary infection). Immunological memory is the basis for vaccination which remains the most successful method for preventing infectious disease. Yet, a fully protective vaccine to prevent any human parasitic disease has not been realized to date. Why is sterilizing immunity to parasitic pathogens so difficulty to achieve? Our previous work on secondary infections with the apicomplexan parasite, Toxoplasma gondii, suggests that parasitic virulence factors are the key to evading adaptive immune responses. Whereas antigenic variation is a major mechanism by which protozoan pathogens such as African Trypanosomes, Plasmodium and Giardia evade B cell-mediated antibody responses, little is known regarding virulence factors that manipulate T cell responses. Given the central role of CD8 T cell-mediated immunity to a wide variety of protozoan pathogens, including T. gondii, this proposal aims to discover parasitic virulence factors that manipulate CD8 T cell responses. Here we report that T. gondii secreted effectors, ROP5 and a second determinant we name ?ROCTR? (Regulator Of CD8 T Cell Responses), work to inhibit T. gondii-specific CD8 T cell responses to parasite-infected cells. ROP5 is a multigene variable family of pseudokinases that are injected into the host cell it invades. Following invasion, T. gondii forms a parasitophorous vacuole (PV) that it replicates within. ROP5 localizes to the cytosolic side of the PV membrane (PVM) protecting it from destruction by the host?s IFN?-induced Immunity Related GTPases (IRGs). Proteins that enter the MHC1 antigen presentation pathway are mainly derived from cytosolic proteins that are degraded by the host?s proteasome. Apicomplexan parasites sequester themselves in a non-fusogenic PV, mostly devoid of host proteins. How does the immune system gain access to vacuolar antigens that are not within conventional endocytic compartments of the cell? Our central hypothesis is that T. gondii ROP5 and an IRG intersect the MHC1 antigen presentation pathway, and that T. gondii ROCTR may regulate this process. However, the identities T. gondii ROCTR and the host IRGs that regulate CD8 T cell activation are unknown. This proposal aims to identify these factors. By analyzing CD8 T cells from T57 mice, which possess a single T cell receptor specificity for the T. gondii TGD05796-103 / Kb antigen complex, T57 T cells were found to secrete IFN? in response to T. gondii-infected macrophages in a strain dependent manner. Using the latest approaches in genome engineering and parasite genetics, we will elucidate mechanisms of immune evasion by T. gondii and determine host requirements for MHC1 antigen presentation of vacuolated pathogens. Through careful dissection, a ?chink in the armor? of T. gondii may be revealed, and give insight into vaccine design for apicomplexan pathogens.